The present invention relates to feedback-resistant homoserine transsuccinylases, to microorganism strains containing these enzymes and to their use for preparing L-methionine or S-adenosylmethionine.
Methionine is an amino acid which is essential for humans and many animals. It is, in particular, produced for the feedstuff market and added to animal feed as the racemate. It is synthesized chemically from acrolein and methanethiol by way of 3-(methylthio)-propionaldehyde, which is converted, with hydrogen cyanide, ammonia and carbon dioxide, into D,L-methionine by way of an hydantoin. The racemate can be resolved enzymically.
S-Adenosylmethionine (SAM) is the most important methyl group donor in metabolism and, in the pharmaceutical field, is used in the treatment of depressions, diseases of the liver and arthritis. Methods which have been described for preparing SAM include, in particular, culturing yeasts (Schlenk F. and DePalma R. E., J. Biol. Chem. 1037-1050 (1957), Shiozaki S. et al., Agric. Biol. Chem. 53, 3269-3274 (1989)) in the presence of the precursor L-methionine and chromatographically purifying after autolysis.
The microbial synthesis of methionine has been investigated particularly intensively in the bacterium E. coli (Greene, R. C., Biosynthesis of Methionine in: Neidhardt F. C., Escherichia coli and Salmonella typhimurium, Cellular and molecular biology, Second Edition, ASM Press, Washington D.C. (1996), pages 542-560 and the references contained therein). It consists of a number of enzyme-catalyzed reactions and is strictly regulated. The first steps in the synthesis, from aspartate to homoserine, proceed in parallel with the formation of the amino acids threonine, leucine, isoleucine and valine. The first step which is specific for the synthesis of methionine is the formation of O-succinylhomoserine from succinyl-CoA and homoserine with the elimination of coenzyme A. This reaction is catalyzed by the enzyme homoserine succinyltransferase (homoserine O-transsuccinylase, MetA, EC 2.3.1.46). SAM is synthesized from L-methionine and ATP in one step.
The activity of homoserine transsuccinylase is inhibited in the presence of L-methionine and/or SAM (Lee L.-W. et al., J. Biol. Chem. 241, 5479-5480 (1966)). While this end product inhibition on the one hand prevents an excessive, energy-consuming synthesis of methionine and SAM in the bacterium, it also, on the other hand, stands in the way of the microbial production of these two substances on an industrial scale. The gene encoding homoserine transsuccinylase consists of 930 base pairs (including the stop codon), while the protein encoded by this gene consists of 309 amino acids. The structure of homoserine transsuccinylase has not thus far been elucidated and it is therefore not possible, either, to identify the amino acids which are involved in an end product inhibition.
A known method of increasing the synthesis of metabolic end products is that of using modified enzymes whose activity can no longer be inhibited by the end product of their metabolic pathway (feedback-resistant mutants). Thus, for example, feedback-resistant mutants of 3-deoxy-D-arabinoheptulonic acid 7-phosphate synthase have been prepared for increasing the synthesis of L-tryptophan and L-phenylalanine (EP0745671A2) and feedback-resistant mutants of chorismate mutase/prephenate dehydratase have been generated for increasing the production of phenylalanine (U.S. Pat. No. 5,120,837).
The E. coli enzyme homoserine transsuccinylase has recently been modified, by mutating the DNA sequence encoding it, such that the activity of the resulting proteins is much less readily inhibited in the presence of 1 mM L-methionine or 1 mM SAM (JP2000139471A). The mutation involved the following amino acid replacements: arginine at position 27 was replaced with cysteine, isoleucine at position 296 was replaced with serine and proline at position 298 was replaced with leucine. The modified homoserine transsuccinylases exhibited residual activities of between 44 and 89% in the presence of 1 mM L-methionine and of between 10 and 73% in the presence of 1 mM SAM. Bacterial strains which contain these modified proteins produce L-methionine to an increased extent. However, in the absence of L-methionine and SAM, these modified homoserine transsuccinylases have an activity which is much less than that seen in the wild type. It is desirable to have available as many variants of homoserine transsuccinylase, which differ in the degree of their activity and in the degree to which they can be inhibited by L-methionine and/or SAM, as possible since the microbial biosynthesis of L-methionine and SAM is highly complex in regard to its course and regulation and, in addition, is interlinked, in a multifaceted manner, with a variety of other metabolic pathways in the cell. It is therefore not possible to make any prediction in advance as to which variant can achieve which effect on the growth of a microorganism strain, on the balance of its vital metabolic processes and on the production of L-methionine and SAM.